We previously demonstrated the occurrence of defective lipid transport and sorting along the endosome/lysosome pathway in a broad collection of sphingolipid storage disease (SLSD) fibroblasts, and in preliminary studies have found that plasma membrane lipid and protein recycling are also significantly perturbed as a result of lipid storage. During the previous grant period we made the exciting finding that over-expression of selected Rab GTPases (rabs 4, 7, or 9) corrects defective membrane traffic in several SLSDs and dramatically reduces accumulation of cholesterol, and perhaps other stored lipids. We hypothesize that the function of selected rab proteins is impaired in SLSDs due to accumulation of SLs and cholesterol. The overall purpose of this grant is to understand how the function of some rab proteins (but not others) is modulated by stored lipids and to develop strategies for overcoming this impaired function. To do so, we will (i) test the hypothesis that stored SLs and cholesterol perturb the mosaic organization of selected rab proteins on endosomes in situ. This will be accomplished by systematically altering cellular lipid composition, by studying lipid microdomains on endosomes of living cells, and by studying the behavior of chimeric rab proteins in response to lipid storage; (ii) perform in vitro studies to determine the mechanism by which stored lipids modulate function of selected rab proteins. These studies will utilize an enriched endosome fraction prepared from normal or SLSD fibroblasts, and will focus on the extraction of prenylated rabs from target membranes by the GDP-dissociation inhibitor. We will determine whether the sensitivity of some (but not other) rabs to stored lipids results from a direct interaction of the lipids with motifs within rab proteins using chimeras of lipid-sensitive and -insensitive rabs; (iii) study the mechanism by which rab protein transduction corrects lipid storage in various SLSD fibroblasts, by quantifying lipid secretion, esterification, or degradation in the treated cells; and (iv) generate a transgenic mouse strain that inducibly over-expresses rab9 in the brain, and cross these mice with an SLSD mouse model to learn whether there is delayed symptom onset and/or increased life span. Together, these studies will provide novel insights into the mechanisms by which lipids and cholesterol regulate membrane trafficking, and may also provide "proof of principle" that modulation of membrane trafficking is a useful approach for treatment of SLSDs.